The coagulation and fibrinolytic cascades comprise a series of zymogen to enzyme conversions which terminate in the proteolytic enzymes thrombin and plasmin, respectively (Mann et al., Ann. N.Y. Acad. Sci. (1991), Vol. 614, pp. 63-75); K. Collen et al., Blood (1991), Vol. 78, pp. 3114-3124; Astrup T., Semin. Thromb. Hemostasis (1991), Vol. 17, pp. 161-174). These enzymes catalyze the deposition and removal of fibrin. A proper balance between the activities of the two cascades is required both to protect the organism from excessive blood loss upon injury and to maintain blood fluidity within the vascular system. Imbalances are characterized by either bleeding or thrombotic tendencies, the latter of which are manifested as, for example, heart attacks and strokes.
Thrombomodulin is a component of the blood vessel wall which binds thrombin and changes its specificity from fibrinogen to protein C, yielding anticoagulant rather than procoagulant activity (Esmon, C. T., FASEB J. (1995), Vol. 9, pp. 946-955). The thrombin-thrombomodulin complex catalyzes cleavage of protein C to activated protein C, which then downregulates the coagulation cascade by proteolytically inactivating the essential cofactors Factor Va and Factor VLLLa (Esmon et al., Ann. N.Y. Acad. Sci. (1991), Vol. 614, pp. 30-43). These events are essential in the regulation of the coagulation cascade.
Early studies suggested that activated protein C is not only an anticoagulant but also profibrinolytic, both in vitro and in vivo (Taylor et al., Thromb. Res. (1985), Vol. 37, pp. 639-649; de Fouw et al., Adv. Exp. Med. Biol., Vol. 281, pp. 235-243). It was later determined that protein C only appears profibrinolytic because it prevents the thrombin-catalyzed activation of a previously unknown fibrinolysis inhibitor, whose precursor has been isolated from plasma and designated TAFI (thrombin-activatable fibrinolysis inhibitor) or PCPB (plasma carboxypeptidase B). The zymogen precursor is activated by thrombin, plasmin or by a thrombin-thrombomodulin complex to produce an enzyme with carboxypeptidase B activity, which inhibits plasminogen activation and thereby prolongs fibrinolysis (Bajzar et al., J. Bio. Chem. (1996), Vol. 270, pp. 14477-14484).
TAFI was discovered independently in three different laboratories. It initially appeared as an unstable carboxypeptidase B-like entity in human serum and was described by Hendricks et al. (Biochim. Biophys. Acta (1990), Vol. 1034, pp. 86-92). Then Eaton et al. (J. Biol. Chem. (1991), Vol. 266, pp. 21833-21838) cloned the cDNA, deduced the amino acid sequence, described its activation by trypsin, and analyzed its enzymatic properties toward synthetic carboxypeptidase B substrates. They designated the protein PCPB, for plasma carboxypeptidase B (see U.S. Pat. No. 5,206,161). Wang et al. (J. Biol. Chem. (1994), Vol. 269, pp. 15937-15944) independently isolated the activated material and named it carboxypeptidase U, where "U" indicates unstable. Nesheim et al (J. Biol. Chem. (1995), Vol. 270, pp. 14477-14484 ) showed that the protein was both activated by thrombin and inhibits fibrinolysis and gave it the name TAFI (thrombin-activatable fibrinolysis inhibitor). Subsequently, Tan and Eaton (Biochemistry (1995), Vol. 34, pp. 5811-5816) studied the trypsin activated enzyme and renamed the protein plasma procarboxypeptidase B (pro-pCPB). The co-identity of TAFI and pro-pCPB (or PCPB, as it was initially designated) has been established by their chromatographic behavior on plasminogen Sepharose and the amino acid sequences present at the activation cleavage site.
Thrombophilia can be defined as a tendency toward venous thromboembolic disease in adults under 50 years old in the absence of known risk factors including, among others, malignancy, immobilization, or major surgery. In principle, a tendency toward venous thrombosis could arise from hyperactive coagulation pathways, hypoactive anticoagulant mechanisms, or hypoactive fibrinolysis. Molecular explanations for some thrombophilic patients have come following the discoveries of hereditary thrombophilia associated with deficiencies of the anticoagulant factors antithrombin III (Egeberg, O., Throm. Diath. Haemorrh. (1965) Vol. 13, p. 516), protein C (Griffin et al., J. Clin. Invest. (1981), Vol. 68, p. 1370), and protein S (Comp et al., N. Engl. J. Med. (1984), Vol. 31, p. 1525). More recently, Dahlback et al. (Proc. Natl. Acad. Sci USA (1993), Vol. 90, p.1004) have identified the presence of a single point mutation in the Factor V gene, which results in the replacement of an amino acid within the activated protein C cleavage site of the Factor Va molecule. The presence of this mutation has been useful in screening the population to determine those at risk for thromboembolic (thrombotic) disease.